The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome.
What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries?
Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS.
In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women.
Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor β (ERβ) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERβ. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability.
AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERβ was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis.
Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7 M (P < 0.05; P < 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. 5α-DHT produced a significant dose-related increase in AMH protein secreted into the media (P = 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERβ produced a corresponding increase in AMH mRNA expression (P = 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P < 0.05, one-way ANOVA) and FSHR (P < 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n = 7, PCOM n = 5, PCOS n = 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P < 0.05, normal n = 6, PCOS n = 3). AMH at 20 and 50 ng/ml decreased KGN-GC cell proliferation but not viability after 8 days of treatment (P < 0.005, two-way ANOVA).
N/A.
Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not DHT.
Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'.
This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose.
N/A.
Dilaver N
，Pellatt L
，Jameson E
，Ogunjimi M
，Bano G
，Homburg R
，D Mason H
，Rice S
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Significance of pro-angiogenic estrogen metabolites in normal follicular development and follicular growth arrest in polycystic ovary syndrome.
Do alterations in pro- and anti-angiogenic estrogen metabolites in follicular fluid (FF) contribute to the follicular growth arrest and anovulation associated with polycystic ovary syndrome (PCOS)?
FF of PCOS women with anovulation have reduced levels of pro-angiogenic estrogen metabolites (EMs) and vascular endothelial growth factor (VEGF) compared to that of fertile women with regular menstrual cycles, but exogenous gonadotropins increase the pro-angiogenic EMs and VEGF levels in PCOS women.
PCOS is characterized by the arrest of follicular development that leads to chronic anovulation. Follicular arrest is generally associated with elevated plasma levels of luteinizing hormone (LH), androgens and anti-Mullerian hormone (AMH). There is also reduced angiogenesis in the follicles of PCOS women compared to those of normal cycling women. It is known that angiogenesis is a critical factor during follicular development. We and other investigators have explored the role of EMs in ovarian angiogenesis, particularly in human corpus luteum function, showing that 4-hydroxyestrone (4-OHE1) and 16-ketoestradiol (16-kE2) have pro-angiogenic effects while 2-methoxyestradiol (2-ME2) and 2-methoxyestrone (2-ME1) have anti-angiogenic effects. Additionally, 2-hydroxyestradiol (2-OHE2), which is produced in the ovary, has proliferative and pro-angiogenic properties. We hypothesized that EMs could be involved in angiogenesis necessary for ovarian follicular development in fertile women, and that dysregulation of these factors may contribute to follicular arrest in PCOS. The relationship between EMs, VEGF and AMH in the pathophysiology of follicular arrest in PCOS has not been previously studied at a follicular level in anovulatory women without ovulation induction.
This is a comparative experimental study of serum and FF collected from different sized follicles (antral ˂10 mm and dominant ˃16 mm) of women with and without ovarian stimulation. The study included women with regular menstrual cycles who were proven to be fertile (n = 20) and PCOS women with follicular arrest who were candidates for ovarian drilling (n = 17), as well as other patients requiring ovarian stimulation, i.e. control women undergoing IVF for male factor infertility (n = 12) and PCOS women undergoing IVF (n = 17). In vitro studies were carried out on granulosa-lutein cells (GCs) obtained from subsets of women undergoing IVF for male factor infertility (n = 6) and PCOS women undergoing IVF (n = 6). GCs were maintained in culture for up to 6 days.
Intrafollicular estradiol, estrone and EMs concentrations were determined by high performance liquid chromatography-mass spectrometry. Testosterone in serum was measured by RIA, and LH, FSH and sex hormone-binding globulin in serum were measured with IRMA kits. AMH was determined in serum and FF by enzyme linked immunosorbant assay (ELISA). VEGF levels were measured in FF and conditioned medium by ELISA. Conditioned medium were obtained from cultured GCs. The angiogenic potential was assessed by in vitro angiogenic assays.
Pro-angiogenic EMs (4-OHE1, 16-kE2 and 2-OHE2) and VEGF were lower in FF of antral follicles of PCOS women with follicular arrest compared those of fertile women with ovulatory cycles (P < 0.05). In contrast, higher concentrations of AMH were found in FF of antral follicles from PCOS women with follicular arrest compared to those of fertile women with ovulatory cycles (P < 0.05). Exogenous gonadotropins used in IVF increased pro-angiogenic EMs and VEGF production in PCOS women, reaching similar profiles compared to control women receiving gonadotropins in their IVF treatment for male factor infertility. The pro-angiogenic EM 2-OHE2 increased the angiogenic potential and VEGF levels of GCs from PCOS women compared to the basal condition (P < 0.05). These findings suggest that there is a role for pro-angiogenic EMs in the control of follicular VEGF production.
The limitations include the possibility that in vitro analysis of GCs might not reflect the in vivo mechanisms involved in the pro-angiogenic action of 2-OHE2 since GCs obtained at the time of oocyte retrieval belong to a very early stage of the luteal phase and might not be representative of GCs during follicular growth. Therefore, our findings do not conclusively rule out the possibility that other in vivo mechanisms also account for defective angiogenesis observed in PCOS.
The present study highlights the significance of EMs, angiogenic factors and AMH and their interaction in the pathophysiology of follicular development in PCOS. This study provides new insights into the role of pro-angiogenic factors in follicular arrest in PCOS.
This study was funded by CONICYT/FONDECYT 1140693 and NIH grant R01HD083323. All authors declare no conflict of interest.
N/A.
Henríquez S
，Kohen P
，Xu X
，Villarroel C
，Muñoz A
，Godoy A
，Strauss JF
，Devoto L
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The phenotypic diversity in per-follicle anti-Müllerian hormone production in polycystic ovary syndrome.
Is intrinsic dysregulation of granulosa cells (GC) and consequent increases in the per-follicle production of anti-Müllerian hormone (AMH), correlated with the phenotypic presentation of women with polycystic ovaries?
Involvement of intrinsic GC dysregulation in oligo-anovulation associated with polycystic ovary syndrome (PCOS) is likely because among women with PCOS, those with oligo-amenorrhea have higher per-follicle AMH production than those who ovulate normally, irrespective of their androgen and/or metabolic status.
Women with PCOS have higher serum AMH level than non-PCOS women due to an increased follicle number and excessive AMH production per follicle, the latter reflecting a putative GC dysfunction that may vary between PCOS phenotypes.
This is a retrospective analysis of data collected from 1021 women undergoing infertility evaluation from March 2011 to October 2013.
The study included women with polycystic ovarian morphology (PCOM) who met the Rotterdam criteria for PCOS (n = 272), women with PCOM only (n = 168) and controls (n = 581).
We used serum AMH to antral follicle count (AFC) ratio (AMH/AFC) as a marker of per-follicle AMH production and checked whether this ratio was associated with the PCOS phenotype and to the menstrual, androgen and metabolic status in women with PCOS, women with PCOM only and in controls. AMH/AFC was significantly higher in oligo-amenorrheic women with PCOS than in eumenorrheic women with PCOS or PCOM (P < 0.001) but also in the latter group compared with controls (P < 0.001) regardless of androgen status. Stepwise discriminant analysis yielded a significant score for the menstrual status with a discriminant power of 26.5% (P < 0.001). This score included AFC, AMH/AFC, waist circumference and LH with partial R(2) of 0.172, 0.042, 0.024 and 0.023, respectively.
The AMH to AFC ratio as a surrogate marker for average AMH may be subject to error because follicles below the sensitivity limit of the ultrasonography used may also contribute to serum AMH concentration and secondly, AFC can be subjective.
The higher AMH/AFC in women with PCOM only than in controls suggests that isolated PCOM may represent a PCOS-like phenotype in which an inherent dysfunction of GC exists but is too mild to affect the ovulatory process.
No funding was obtained for this study. There are no conflicts of interest to be declared.
Non-applicable.
Alebić MŠ
，Stojanović N
，Duhamel A
，Dewailly D
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